This paper reviews the current status of our understanding of resistance mechanisms of three major classes of antifungal drugs for systemic use, amphotericin B (AMPH), flucytosine (5-FC) and several azole antifungals, in particular fluconazole (FLCZ), at the molecular and cellular levels. Although the number of reports of AMPH- or 5-FC-resistant fungal species and strains is limited, several mechanisms of resistance have been described. AMPH-resistant Candida have a marked decrease in ergosterol content compared with AMPH-susceptible control isolates. A lesion in the UMP-pyrophosphorylase is the most frequent determinant of 5-FC resistance in C. albicans. Recently resistance of C. albicans to azoles has become an increasing problem. Extensive biochemical studies have highlighted a significant diversity in mechanisms conferring resistance to FLCZ and other azoles, which include alterations in sterol biosynthesis, target site, uptake and efflux. Among them, the most important mechanism clinically is reduced access of the drug to the intracellular P450_14DM target, probably because of the action of a multidrug resistance efflux pump, and overproduction of that target. However, other possible resistance mechanisms for azoles remain to be identified.

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We compared the in vitro and in vivo antifungal activities of D0870, a new triazole antifungal agent, with those of other antifungal agents against 8 clinical isolates of fluconazole-resistant Candida albicans. Microdilution testing was performed according to National Committee for Clinical Laboratory Standards (NCCLS) document M27-T. Minimal inhibitory concentration of D0870 (≤0.004-1.0μg/ml) was lower than those of fluconazole (2->64μg/ml) and itraconazole (0.031-8.0μg/ml). In systemic infection models with C. albicans in normal and immunosuppressed mice, D0870 at 0.3-30mg/kg/day for 5 days after infection prolonged survival of the animals and showed the highest efficacy among the triazole antifungal agents. At pH 7 and 37°C in Sabouraud dextrose broth (SDB), D0870 inhibited the growth of C. albicans and acted cytocidally against one of the middle-resistant strains. In an in vivo study against this strain, D0870 at 10mg/kg/day for 5 days after infection significantly reduced kidney colony counts (2850±406→997±537 CFU/kidney, P<0.05) on day 7 after infection in comparison with those of the control mice at 24h after infection. Plasma concentration of D0870 after a single oral administration at 10mg/kg maintained a sufficient level for interpretation of in vivo antifungal activities.
These results suggest that D0870 has strong antifungal activities against clinical isolates of fluconazole-resistant C. albicans in vitro and in vivo, and that these strong activities are at least partially concerned with the fungicidal action.

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We examined the effects of Asp-hemolysin from Aspergillus fumigatus Fresenius-Muramatsu strain on the viability and cytokine gene expression of mouse peritoneal macrophages (Mφ). The cytotoxic activity of Asp-hemolysin to Mφ cultured in FCS-RPMI medium was increased in a dose-dependent manner. Treatment of Asp-hemolysin with N-ethylmaleimide or sulfo-N-hydroxy-sulfosuccinimide-acetate caused a remarkable loss of the cytotoxic activity, however, the cytotoxic activity of Asp-hemolysin to Mφ cultured in serum-free medium was significantly increased as compared with that in FCS-RPMI medium. As other biological activities of Asp-hemolysin, tumor necrosis factor-α (TNF-α), interleukin-1α (IL-1α) and interleukin-1β (IL-1β) mRNA expression were observed in Mφ cultured with 1μg/ml of Asp-hemolysin.

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Clinical evaluation was retrospectively made of the results of serological diagnostic methods using plasma and/or sera of patients for the diagnosis of aspergillosis, candidosis, and pneumocystosis. Specimens were drawn from 8 patients with invasive aspergillosis, 3 with aspergilloma, 9 with candidosis, 4 with pneumocystosis, and 15 with no fungal infections.
In invasive aspergillosis, the sensitivities of the (1→3)-β-D-glucan measurement test using chromogenic and turbidimetric methods were 78.6% and 82.1%, with specificities of 75% and 87.5%, respectively. The sensitivity of the Pastorex Aspergillus test for invasive aspergillosis was 16.7%, with a specificity of 92.3%. In candidosis, the sensitivities of the (1→3)-β-D-glucan test using the above two methods were 84.2% and 100%, with specificities of 75% and 87.5%, respectively. The sensitivity of the CAND-TEC test and the Pastorex Candida test for candidosis were 68.8% and 16.7%, with specificities of 57.1% and 100%, respectively.
These results indicate that the (1→3)-β-D-glucan measurement methods are more reliable in clinical application than the other antigen detection methods, but they still lack efficiency in differentiating fungal infections such as aspergillosis, candidosis and pneumocystosis. For a more exact diagnosis of systemic fungal infections, detailed studies on the clinical symptoms are considered essential.

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We report a case of a healthy 61-year-old woman with discoloration of the nail on her right big toe. We first treated her with topical steroid and urea under suspected diagnosis of nail eczema, but the lesion remained. In culture, black, shiny, pasty and yeast-like colonies grew repeatedly. Examination of debris from her nail showed dematiaceous spherical cells and hyphal elements. Microscopically, annelloconidia were produced at the apical ends of anellidic conidiogenous cells. This colony grew at 40°C. Mitochondrial DNA restriction fragment length polymorphism was analysed in this strain and its restriction pattern confirmed the isolate to be Exophiala dermatitidis. Based on these findings, we diagnosed this nail deformity as fungal melanonychia due to Exophiala dermatitidis. This is the third reported case of this disease.

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The effect of elastolytic proteinase on quantitative nitroblue tetrazolium (NBT) dye reduction and chemotaxis of human neutrophil was examined. Elastolytic proteinase is derived from Aspergillus fumigatus 9409. NBT dye reduction was inhibited significantly by 133μg/ml of elastolytic proteinase (p<0.05). Chemotaxis was also inhibited significantly by 400μg/ml of the enzyme, compared to the control group (p<0.05). These findings suggest that elastolytic proteinase, depending on its concentration, acts as an inhibitory agent to both NBT dye reduction and chemotaxis activities in the human neutrophil. The findings that this enzyme suppressed neutrophil function in A. fumigatus infection is of importance, because it is now suspected that the enzyme has the potential to cause infection.

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